Analytical methods for protein kinase profiling by using fluorescent Phos-tag molecules

• Project/Area Number: 22390006
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Hiroshima University
• Principal Investigator: KOIKE Tohru 広島大学, 医歯薬保健学研究院, 教授 (90186586)
• Project Period (FY): 2010-04-01 – 2014-03-31
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥16,510,000 (Direct Cost: ¥12,700,000、Indirect Cost: ¥3,810,000); Fiscal Year 2013: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2012: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2011: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2010: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000)
• Keywords: プロテオミクス / キナーゼ / フォスファターゼ / 蛍光分析 / ケミカルバイオロジー / リン酸化タンパク質 / 蛍光分析法 / 磁気ビーズ / キナーゼ反応 / シグナル伝達 / タンパク質リン酸化 / タンパク質 / リン酸化反応

Research Abstract

By utilizing the Phos-tag molecule and its fluorescent derivatives, we have developed convenient and reliable methods for the analysis of phosphorylated biomolecules such as peptides and proteins. In this program, we applied the Phos-tag technology to (1) FRET system for kinase/phosphatase profiling using Phos-tag, (2) fluorometric analysis of phosphorylated proteins, (3) analysis of phosphorylation status of multi-phosphorylated biomolecules. We believe that our Phos-tag technology will result in great progress in phosphoproteomics.

Research Products

• [Journal Article] Profiling of Protein Thiophosphorylation Using Phos-tag Affinity Electrophoresis: Evaluation of Adenosine 5′-O-(3-thiotriphosphate) as a Phosphoryl Donor in Protein Kinase Reactions (2014)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, A. Shiba, K. Edahiro, Y. Inoue, K. Yamamoto, and T. Koike
  • Journal Title: Proteomics Volume: 14 Issue: 6 Pages: 668-679
  • DOI: 10.1002/pmic.201300533
  • Peer Reviewed
• [Journal Article] Sandwich assay for phosphorylation of protein multiplexes by using antibodies and Phos-tag (2013)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • Journal Title: Analytical Biochemistry Volume: 438 Issue: 2 Pages: 104-106
  • DOI: 10.1016/j.ab.2013.03.029
  • Peer Reviewed
• [Journal Article] A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules (2013)
  • Author(s): M. Tsunehiro, Y. Meki, K. Matsuoka, E. Kinoshita-Kikuta, E. Kinoshita and T. Koike
  • Journal Title: Journal of Chromatography B Volume: 925 Pages: 86-94
  • DOI: 10.1016/j.jchromb.2013.02.039
  • Peer Reviewed
• [Journal Article] Phos-tag-Based Microarray Techniques Advance Phosphoproteomics (2013)
  • Author(s): Eiji Kinoshita, Emiko Kinoshita-Kikuta and Tohru Koike
  • Journal Title: Journal of Proteomics & Bioinformatics Volume: S6 Issue: S6 Pages: 8-8
  • DOI: 10.4172/jpb.s6-008
  • Peer Reviewed
• [Journal Article] A Laborsaving, Timesaving, and More Reliable Strategy for Separation of Low-Molecular-Mass Phosphoproteins in Phos-tag Affinity Electrophoresis (2012)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: International Journal of Chemistry Volume: 4 Issue: 5 Pages: 1-8
  • DOI: 10.5539/ijc.v4n5p1
  • Peer Reviewed
• [Journal Article] Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin (2012)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, Y. Sugiyama, Y. Fukada, T. Ozeki, and T. Koike
  • Journal Title: Proteomics Volume: 12 Issue: 7 Pages: 932-937
  • DOI: 10.1002/pmic.201100639
  • Peer Reviewed
• [Journal Article] Separation and identification of four distinct serine-phosphorylation states of ovalbumin by Phos-tag affinity electrophoresis (2012)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: Electrophoresis Volume: 33 Issue: 5 Pages: 849-855
  • DOI: 10.1002/elps.201100518
  • Peer Reviewed
• [Journal Article] Phos-tag SDS-PAGE systems for phosphorylation profiling of proteins with a wide range of molecular masses under neutral pH conditions (2012)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • Journal Title: Proteomics Volume: 12 Issue: 2 Pages: 192-202
  • DOI: 10.1002/pmic.201100524
  • Peer Reviewed
• [Journal Article] A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the kinase reaction of a substrate peptide (2011)
  • Author(s): M. Somura, K. Takiyama, E. Kinoshita- Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: Analytical Methods Volume: 3 Issue: 6 Pages: 1303-1309
  • DOI: 10.1039/c1ay05016h
  • Peer Reviewed
• [Journal Article] Zinc(II)-cyclen polyacrylamide gel electrophoresis for detection of mutations in short Ade/Thy-rich DNA fragments (2011)
  • Author(s): Eiji Kinoshita
  • Journal Title: Analytical Biochemistry Volume: 408 Pages: 348-350
  • Peer Reviewed
• [Journal Article] A novel phosphate-affinity bead immobilized with Phos-tag for separation and enrichment of phosphopeptides and phosphoproteins (2011)
  • Author(s): Emiko Kinoshita-Kikuta
  • Journal Title: Journal of Integrated Omics Volume: 1 Pages: 157-169
  • Peer Reviewed
• [Journal Article] The DNA-binding activity of mouse DNA methyltransferase 1 is regulated by phosphorylation with casein kinase 1delta/ epsilon (2010)
  • Author(s): Sugiyama Y, Hatano N, Sueyoshi N, Suetake I, Tajima S, Kinoshita E, Kinoshita-Kikuta E, Koike T, and Kameshita I
  • Journal Title: Biochem J Volume: 427 Issue: 3 Pages: 489-497
  • DOI: 10.1042/bj20091856
  • Peer Reviewed
• [Journal Article] A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag (2010)
  • Author(s): J. Morishige, M. Urikura, H. Takagi, K. Hirano, T. Koike, T. Tanaka & K.
  • Journal Title: Satouchi Rapid Commun. Mass Spectrom Volume: 24 Issue: 7 Pages: 1075-1084
  • DOI: 10.1002/rcm.4484
  • Peer Reviewed
• [Journal Article] The DNA-binding activity of mouse DNA methyltransferase 1 is ragulated phosphorylation with casein kinase 1σ/ε (2010)
  • Author(s): Yasunori Sugiyama
  • Journal Title: Biochemical Journal Volume: 427 Pages: 489-497
  • Peer Reviewed
• [Presentation] 迅速かつ簡便にリン酸化生体分子を分離精製するためのPhos-tag磁気ビーズの開発 (2013)
  • Author(s): 常弘昌弥, 目木勇馬, 木下恵美子, 木下 英司, 小池 透
  • Organizer: 第64回日本電気泳動学会
  • Place of Presentation: 仙台
  • Year and Date: 2013-11-15
• [Presentation] Phos-tag Biotin as an on-demand tool for study on protein phosphorylome (2013)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • Organizer: 12th HUPO Annual World Congress
  • Place of Presentation: Yokohama
• [Presentation] 中性 phos-tag PAGEを用いたタンパク質リン酸化反応の解析 (2012)
  • Author(s): 木下英司, 木下恵美子, 小池 透
  • Organizer: 第85回日本生化学会大会
  • Place of Presentation: 福岡
  • Year and Date: 2012-12-15
• [Presentation] Phos-tag Biotinの開発とリン酸化プロ テオミクスへの応用 (2012)
  • Author(s): 木下英司, 木下恵美子, 小池 透
  • Organizer: 10th JHUPO
  • Place of Presentation: 東京
  • Year and Date: 2012-07-27
• [Presentation] Phos-tag SDS-PAGE systems for protein phosphorylation profiling under neutral pH (2012)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • Organizer: 11th HUPO Annual World Congress
  • Place of Presentation: Boston, USA
• [Presentation] 改良型Phos-tag Biotinを用いたペプチドマイクロアレイ解析によるリン酸化反応プロファイリング (2011)
  • Author(s): 木下英司, 木下恵美子, 小池 透
  • Organizer: 9th JHUPO
  • Place of Presentation: 新潟
  • Year and Date: 2011-07-28
• [Presentation] リン酸基結合タグ分子(Phos-tag)を用いたリン酸化生体分子の解析法 (2011)
  • Author(s): 小池 透
  • Organizer: 生体機能関連化学部会 若手の会 第23回サマースクール
  • Place of Presentation: 廿日市市
  • Year and Date: 2011-07-23
• [Presentation] リン酸基結合タグ分子(Phos-tag)を用いたリン酸化生体分子の解析法 (2011)
  • Author(s): 小池透
  • Organizer: 日本化学会生体関連化学部会サマースクール
  • Place of Presentation: 広島県廿日市市(招待講演)
  • Year and Date: 2011-07-23
• [Presentation] Phos-tag-based fluorescence resonance energy transfer system for the analysis of phosphorylated molecules (2010)
  • Author(s): Masao Somura
  • Organizer: 環太平洋国際化学会 PACIFICHEM 2010
  • Place of Presentation: 米国ハワイ州ホノルル
  • Year and Date: 2010-12-16
• [Presentation] Phos-tag fluorescence resonance energy transfer system for the analysis of phosphorylated molecules (2010)
  • Author(s): M. Somura, K. Takiyama, T. Nagai, E. Kinoshita, E. Kinoshita-Kikuta, and T. Koike
  • Organizer: International Chemical Congress of Pacific Basin
  • Place of Presentation: Honolulu, USA
• [Presentation] 近接Phos-tagゲルを用いたリン酸化タンパク質の電気泳動法
  • Author(s): 目木 勇馬，木下 英司，木下 恵美子, 小池 透
  • Organizer: 日本電気泳動学会
  • Place of Presentation: 仙台市
• [Presentation] 迅速かつ簡便にリン酸化生体分子を分離精製するためのPhos-tag磁気ビーズの開発
  • Author(s): 常弘 昌弥, 目木 勇馬, 木下 恵美子，木下 英司，小池 透
  • Organizer: 日本電気泳動学会
  • Place of Presentation: 仙台市
• [Presentation] Quantitative analysis of effects of pH on the stability of His- and Asp-phosphoproteins in bacterial two-component system by using phosphate-affinity SDS-PAGE
  • Author(s): Akio Shiba, Keisuke Edahiro, Emiko Kinoshita-Kikuta, Eiji Kinoshita, and Tohru Koike
  • Organizer: 12th HUPO World Congress
  • Place of Presentation: 横浜市
• [Presentation] Quantitative analysis of the phosphorylation state of a hybrid histidine kinase in bacterial two-component system by using phosphate-affinity SDS-PAGE
  • Author(s): Keisuke Edahiro, Akio Shiba, Emiko Kinoshita-Kikuta, Yuki Inoue, Eiji Kinoshita, and Tohru Koike
  • Organizer: 12th HUPO World Congress
  • Place of Presentation: 横浜市
• [Presentation] Phos-tag SDS-PAGE systems for protein phosphorylation profiling under neutral pH conditions
  • Author(s): Eiji Kinoshita
  • Organizer: HUPO2012 conference
  • Place of Presentation: Boston, USA
• [Book] Phos-tag Affinity Electrophoresis for Protein Kinase Profiling "Protein Kinase Technologies, Neuromethods" (2012)
  • Author(s): E. Kinoshita, E. Kinoshita-Kikuta, T. Koike
  • Publisher: Humana Press, Springer
• [Book] Phos-tag ゲルによるリン酸化タンパク質の分離・同定法,臨床プロテオーム(日本 プロテオーム研究会編) (2012)
  • Author(s): 木下英司, 木下恵美子, 小池 透
  • Publisher: 金原出版
• [Book] 蛍光性Phos-tag を用いたタンパク質リ ン酸化反応の解析法,生物物理化学, 日本電気泳動学会編 (2012)
  • Author(s): 木下英司, 木下恵美子, 多幾山敬, 宗村雅男, 小池 透
• [Book] 特集リン酸化生体分子解析のためのフォスタグテクノロジー (2012)
  • Author(s): 木下英司
  • Total Pages: 75
  • Publisher: 日本泳動学会
• [Remarks]
  • URL: http://home.hiroshima-u.ac.jp/tkoike/
• [Remarks] 医薬分子機能科学研究室
  • URL: http://home.hiroshima-u.ac.jp/tkoike/index.html
• [Remarks] 医薬分子機能科学研究室
  • URL: http://home.hiroshima-u.ac.jp/tkoike/index.html
• [Remarks]
  • URL: http://home.hiroshima-u.ac.jp/tkoike/
• [Remarks]
  • URL: http://home.hiroshima-u.ac.jp/tkoike/